Pregnancy-associated endometrial alpha 1-globulin (alpha 1-PEG) is quantitatively the major secretory protein product, synthesized and secreted in vitro, of the human decidualized endometrium during pregnancy. This protein has been purified from cytosolic extracts of this tissue and has now been characterized as a 32 kDa somatomedin/insulin-like growth factor (IGF)-binding protein. Immunoreactive alpha 1-PEG isolated from amniotic fluid exhibited identical physiochemical properties and IGF-I-binding characteristics. In cytosolic extracts of pregnancy endometrium, in incubation medium of this tissue and in amniotic fluid, the 32 kDa protein represented the major alpha 1-PEG immunoreactive protein and major IGF-I-binding component. Purified alpha 1-PEG and incubation medium of pregnancy endometrium competed for IGF-I with placental membrane IGF receptors in vitro. The implications of the endometrial source of IGF-I-binding protein are discussed with reference to the origin of the amniotic fluid and serum small Mr IGF-binding protein and to the suggested paracrine effect upon trophoblast proliferation.

The major secretory product of the baboon and human decidua during pregnancy is an insulin-like growth factor-binding protein (IGF-BP). This study was designed to determine the site and regulation of synthesis of this protein in the nonpregnant baboon based on our previous findings that this molecule is biochemically and immunologically similar in the two species during pregnancy. Endometrial tissue was obtained from cycling baboons and steroid-treated ovariectomized animals. Portions of the tissue were fixed for immunocytochemical analysis, cultured in the presence or absence of cyclohexamide and actinomycin-D, or snap-frozen in liquid nitrogen for RNA isolation. Immunostaining using monoclonal antibodies to IGF-BP indicated that the protein was localized predominantly in the epithelium of the deep glands and was most intense during the late luteal stage of the cycle. Immunoreactive product was also present in tissue from estrogen-primed progesterone-treated ovariectomized animals. However, the staining pattern was more variable and less intense than that in intact animals. Western blots of explant culture media showed the presence of an immunoreactive product only in those tissues that also demonstrated immunocytochemical localization. The absence of an immunoreactive band in medium obtained from tissue incubated in the presence of cyclohexamide suggested that this protein was synthesized de novo. The mRNA coding for IGF-BP appeared to be stable, as synthesis in explant cultures continued in the presence of actinomycin-D. The cDNA probe hybridized to a single message transcript of 1.65 kilobases. The presence of mRNA in tissues coding for this protein correlates with the immunochemical data relating to the site and hormonal regulation of its synthesis. The presence of this protein in the glandular epithelium of the baboon endometrium may have implications in the autocrine and/or paracrine regulation of trophoblast growth and penetration during implantation.

A chick non‐muscle α‐actinin cDNA probe encoding the EF‐hand region of molecule was used to screen a λgt10 chick brain cDNA library from 14‐day embryos. A partial 2.1‐kb α‐actinin cDNA was isolated (8W cDNA) which encoded a protein identical to chick skeletal‐muscle α‐actinin, except in the C‐terminal part of the first EF hand. In the variant, the 22 residues found in the skeletal‐muscle isoform were replaced by a stretch of 26 unique residues. Analysis of the structure of the skeletal‐muscle α‐actinin gene showed that the region of divergence was encoded by two exons which are alternatively spliced. Quantitative reverse transcriptase/polymerase chain reaction (RT/PCR) was used to investigate the levels of the α‐actinin transcripts in various tissues. The skeletal‐muscle α‐actinin variant was expressed at low levels in brain, liver and spleen, but could not be detected in skeletal muscle. Surprisingly, skeletal‐muscle α‐actinin mRNA was also expressed in brain, liver and spleen. The RT/PCR products were authenticated by using diagnostic restriction enzyme sites and by sequencing. The splice variant derived from the skeletal‐muscle α‐actinin gene was also detected in a variety of cDNA libraries from both adult and embryonic tissues by PCR. Although a transcript encoding this α‐actinin splice variant is expressed in non‐muscle tissues, neither of the two EF‐hands would be predicted to be functional, making it unlikely to be a typical non‐muscle isoform which are calcium‐sensitive with respect to binding actin. The two vertebrate non‐muscle α‐actinins sequenced to date also have a spacer of five amino acids between the two EF hands, whereas in the variant, the spacer is just four residues in length. Further analysis will be required before this α‐actinin isoform, which we refer to as SKv, can be classified as muscle or non‐muscle α‐actinin. We propose a new nomenclature to describe the various α‐actinin genes and their transcripts.

The major secreted protein of the human decidua (pregnancy-associated endometrial alpha 1-globulin [alpha 1-PEG]), is an insulin-like growth factor-binding protein (IGF-BP) that is immunologically and biochemically similar to placental protein 12 (PP12) extracted from term human placenta. Since previous studies have demonstrated that the baboon and human endometrium synthesize and release a number of biochemically and immunologically related polypeptides in culture, this study was undertaken to further characterize a related IGF-BP in baboon placental tissues. Decidua, chorio-amniotic membranes with adhering decidua (CAM-D), and placental villi were obtained from pregnant baboons between Days 134 and 160 of gestation by Cesarean sections. Portions of tissue were either cultured in the presence of 35S-methionine, fixed for immunocytochemistry, or frozen in liquid nitrogen for cytosol extraction. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of tissue culture media (TCM) revealed that the major secretory product of the decidua and CAM-D was an acidic polypeptide (Mr 33,000). Western blot analysis and immunoprecipitation of TCM with murine monoclonal antibody (B2H10) against human alpha 1-PEG demonstrated that this molecule, secreted by the baboon decidua and CAM-D, but not the placental villi, was immunologically identical to the human IGF-BP. Immunocytochemical localization of IGF-BP was intense in the cytoplasm of stromal cells in decidua and CAM-D and absent in the placenta. Gel filtration of TCM and cytosol followed by screening of eluates for 125I-IGF-I binding resolved two peaks (Mr greater than 100,000 and 35,000) of specific IGF-BP in decidua and CAM-D. The 35,000 peak had 100-200 times the binding capacity of the Mr greater than 100,000 peak and a Kd of 1.14-1.83 nM. The eluates contained in the Mr 35,000 peak were also immunoreactive to alpha 1-PEG, as accessed by a polyclonal radioimmunoassay. Affinity cross-linking with 125I-IGF-I followed by sodium dodecyl sulfate-PAGE revealed an immunoreactive complex of Mr 36,000, confirming that the baboon protein represents a high affinity IGF-BP. These studies indicate that the hypertrophied stromal cells of the baboon decidua and CAM-D synthesize and release an IGF-BP as their major secretory product, analogous to the situation in humans. The results of this study suggest that this protein may play a role in the regulation of IGF action during pregnancy.

Pregnancy-associated endometrial alpha 1-globulin (alpha 1-PEG), a 29,000 mol wt insulin-like growth factor-binding protein, is the major secretory protein of the human endometrium from the latter half of the first trimester to the end of pregnancy. It is also produced and secreted by nonpregnant endometrial tissue, especially during the late secretory phase of the menstrual cycle. We studied the localization of this protein in the endometrium during different phases of the cycle using immunohistochemistry with monoclonal antibodies. Immunostaining was first observed in the midsecretory phase and was most consistently detected during the late secretory phase, during which it was principally associated with stromal cell populations. These cells were localized initially surrounding spiral arteries and subsequently also in the subluminal epithelial region of the endometrium. These results suggest that alpha 1-PEG synthesis is principally associated with the process of stromal cell differentiation. i.e. predecidualization. However, the presence of alpha 1-PEG was not directly correlated with the presence of mature predecidual cells, suggesting that its synthesis is not an inherent feature of this cell.